Identification of small, noncoding RNAs[unreadable] [unreadable] We have been carrying out several different systematic screens for small, noncoding RNA genes in E. coli. These screens are all applicable to other organisms. One approach based on computer searches of intergenic regions for extended regions of conservation among closely related species led to the identification of 17 conserved small RNAs. Another screen for small RNAs that coimmunoprecipitate with the RNA binding protein Hfq allowed us to detect six less well conserved RNAs. A third approach of size fraction of total RNA followed by linker ligation and cDNA synthesis resulted in the identification of still other small RNAs. We have recently obtained tiled microarrays which provide coverage of the entire E. coli genome and are using these arrays to extend our identification of the small RNAs.[unreadable] [unreadable] [unreadable] Development of general approaches for the characterization of small, noncoding RNAs[unreadable] [unreadable] We also have been developing tools for the characterization of small RNA regulators. Detection of RNAs on microarrays has become a standard approach for molecular biologists. However, current methods frequently discriminate against structured andor small RNA species. In collaboration with Dr. Gottesman and Dr. Leppla, we developed an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNARNA hybrid-specific mouse monoclonal antibody. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of E. coli much more efficiently than the commonly-used cDNA-based method. [unreadable] [unreadable] Many bacterial sRNAs act as post-transcriptional regulators by basepairing with target mRNAs. While the number of characterized small RNAs has steadily increased, only a limited number of the corresponding mRNA targets have been identified. In collaboration with Dr. Gottesman and Dr. Tjaden, we developed and tested a program, TargetRNA, that predicts the targets of these small RNA regulators. The program was evaluated by assessing whether previously known targets could be identified. The program was then used to predict targets for the E. coli RNAs RyhB, OmrA, OmrB and OxyS, and the predictions were compared with changes in whole genome expression patterns observed upon expression of the small RNAs. [unreadable] [unreadable] [unreadable] Characterization of specific small, noncoding RNAs[unreadable] [unreadable] A growing focus of the group has been to elucidate the functions of the small RNAs in E. coli. We previously showed that OxyS RNA, whose expression is induced by OxyR in response to oxidative stress, acts to repress translation by basepairing with target mRNAs. OxyS RNA action is dependent on the Sm-like Hfq protein, which functions as a chaperone to facilitate OxyS RNA basepairing with its target mRNAs. We also discovered that the abundant 6S RNA binds and modifies RNA polymerase. In addition, we elucidated the functions of two other small RNAs, the MicC RNA and the GadY, that also bind Hfq and act by basepairing. We found the MicC RNA represses translation of the OmpC outer membrane porin. Interestingly, under most conditions, the MicC RNA shows expression opposite that of the MicF RNA, which represses expression of the OmpF porin. Basepairing between the GadY RNA and the 3-untranslated region (3 UTR) of the gadX mRNA encoded opposite gadY leads to increased levels of the gadX mRNA and GadX protein. Increased GadX levels in turn result in increased expression of the acid-response genes controlled by the GadX transcription factor.[unreadable] [unreadable] Recently, we characterized a small RNA (SymR) is encoded in cis to an SOS-induced gene whose product shows homology to the antitoxin MazE (SymE). We showed that synthesis of the SymE protein is tightly repressed at multiple levels by the LexA repressor, the SymR RNA and the Lon protease. SymE co-purifies with ribosomes and overproduction of the protein leads to cell growth inhibition, decreased protein synthesis and increased RNA degradation. These properties are shared with several RNA endonuclease toxins of the toxin-antitoxin modules, and we reported that the SymE protein represents evolution of a toxin from the AbrB fold, whose representatives are typically antitoxins. We suggest that SymE promotion of RNA cleavage may be important for the recycling of RNAs damaged under SOS-inducing conditions. Studies to further characterize the OxyS, GadY and SymR RNAs and to elucidate the roles of other newly-discovered small RNAs are ongoing.[unreadable] [unreadable] [unreadable] Characterization of small ORFs[unreadable] [unreadable] In our genome-wide screens for small RNAs, we found that a number of short RNAs actually encode small proteins. Although small proteins have largely been missed, the few small proteins that have been studied in detail in bacterial and mammalian cells have been shown to have important functions in signaling and in cellular defenses. Thus we established a project to identify E. coli proteins of less than 50 amino acids and elucidate their functions using many of the approaches the group has used to characterize the functions of small, noncoding RNAs.[unreadable] [unreadable] [unreadable] Characterization of the OxyR and Fur transcription regulators[unreadable] [unreadable] Previously, a major focus of the laboratory was the characterization of the OxyR transcription regulator, its sensitivity to oxidation and its binding to DNA. In the past year, we also completed a long standing study of OyxR mutants to define a region where OxyR contacts RNA polymerase. In collaboration with Thomas Schneider, we also completed a computational analysis of DNA binding sites for the iron repressor protein Fur.